Alpha-methylacyl-CoA racemase (AMACR) is a well-characterized enzyme which plays a key role in peroxisomal β-oxidation of dietary branched-chain fatty acids and C27-bile acid intermediates. Ferdinandusse et al., J. Lipid Res. 41:1890–1896 (2000). AMACR catalyzes the conversion of (R)-α-methyl-branched-chain fatty acyl-CoA esters to their (S)-stereoisomers. Cuebas et al., Biochem. J. 363:801–807 (2002); Schmitz et al., Eur. J. Biochem. 231:815–822 (1995). AMACR was isolated from human liver as a 47-kDa monomer and is mainly localized in peroxisomes with a small amount detected in the mitochondria. Schmitz et al., Eur. J. Biochem. 231:815–822 (1995); Schmitz et al., Eur. J. Biochem. 222:313–23 (1994). The isoelectric point for AMACR isolated from human liver is pH 6.1, and the enzyme is optimally active between pH 7 and pH 8.
Elevated expression of AMACR has been shown to be a biomarker for several types of cancer such as prostate, colorectal, ovarian, breast, bladder, lung, renal cell carcinoma, lymphoma, and melanoma. Rubin et al., JAMA 287: 1662–1670 (2002); Luo et al., Cancer Res. 62:2220–2226 (2002); Sreekumar et al., J. Natl. Cancer Inst. 96:834–843 (2004). Studies also show that alpha-methylacyl-CoA racemase enzymatic activity is elevated in prostate cancer tissue specimens. Kumar-Sinha et al., Am. J. Pathol. 164:787–93 (2004). PCT WO 02/27324 and U.S. Appl. Pub. 2002/0123081 describes methods for identifying patients having or at risk of developing prostate cancer and patients having or at risk of developing a cancer arising from metastasis of a prostate cancer to another tissue by measuring the expression or activity of AMACR.
Several methods have been reported for assaying enzymatic activity of AMACR. Schmitz et al. (Eur. J. Biochem. 222:313–23 (1994)) describe a radiometric assay for AMACR enzymatic activity, in which 2-methyl[2-(3)H]acyl-CoAs is used as substrate. Veldhoven et al. (Biochem. Biophys. Acta 1347:62–68 (1997)) describe an alternative enzymatic assay for AMACR, in which AMACR is combined with 2R-methyl-pentadecanoyl-CoA. The reaction product, a 2S-isomer, is desaturated by an excess of added oxidase (pristanoyl CoA oxidase) resulting in the production of hydrogen peroxide, which is monitored by means of peroxidase in the presence of a suitable hydrogen donor.
However, there is still a need for a reliable and sensitive method for assaying AMACR enzymatic activity in a sample (e.g., for diagnosing cancer).